The present invention relates to a method of and apparatus for measuring a fluorescence decay time.
Since all electrons are normally paired in a molecule, a ground state of a molecule is usually a singlet state. If a molecule in a singlet ground state undergoes a strong initial absorption of a photon to an upper state which is also a singlet state, the excitation energies may be released subsequently by a process which includes the emission of a less energetic photon, i.e. by fluorescence. A more detailed description of the mechanism of fluorescence is given at pages 75 to 78 of xe2x80x9cQuanta a handbook of conceptsxe2x80x9d by P. W. Atkins, Clarendon Press, Oxford, 1974 (ISBN 019 855 494X). In the present specification, an exciting photon may be referred to as light, but it will be understood that the term light is being used in a general sense and is not limited to photons of visible light. For example, the exciting light photons may be infra-red or ultra-violet.
Fluorescent dye molecules are known to be useful for labelling materials, especially biological materials, to enable the labelled materials to be detected in small quantities or distinguished in mixtures. One known technique is that of single molecule detection (SMD) by time correlated single photon counting (TCSPC) in which a single molecule of a fluorophore is repeatedly stimulated to fluorescence by being struck by a regular train of up to 10,000 pulses of light of the appropriate exciting wavelength, measurement of the time between irradiation with an exciting pulse and emission of a fluorescence photon, and determination of the fluorescence decay time from a histogram of the measured times.
A problem of this conventional procedure is that a significantly long time is required to fire the necessary number of exciting pulses at a fluorophore molecule.
Accordingly, the present invention provides a method and apparatus as defined hereinafter in the claims.
In a preferred embodiment of the invention, laser pulses of a selected wavelength are fired from a laser head through an objective lens to a focal spot in a sample spot. If a target fluorophore molecule is present within the focal spot and emits a fluorescence photon which is collected by the objective lens, the fluorescence photon is reflected by a dichroic mirror and passes through a filter, which blocks photons of other wavelengths, to a single photon counting photomultiplier unit. On detecting a fluorescence photon, the photomultiplier unit generates a detection output pulse which is coupled, with a short delay, by a pulse controller to a driver circuit which in response thereto causes the laser head to generate another laser pulse, so that the interval between the laser pulses is substantially equal to the time between excitation of and fluorescence emission by the fluorophore molecule. A personal computer coupled to the photomultiplier unit and the driver circuit determines the fluorescence decay time by measurement of a sufficiently large number of fluorescence events. The sample spot is a disk-like spot of a liquid medium containing a volume density of target fluorophore molecule such that there will most probably be one target fluorphore molecule in the volume of the medium through which a laser pulse passes, the latter volume depending upon the thickness of the sample spot, the diameter of the focal spot, and the numerical aperture of the objective lens. The sample spot is supported by a sample slide. Preferably the sample slide supports a rectangular array of sample spots and is itself secured to a stage movable in steps in two orthogonal directions, to which the rows and columns of the array of sample spots are respectively parallel, so that the array of sample spots can be in effect scanned by the focal spot defined by the objective lens. The personal computer controls movement of the stage and sets a maximum N for the number of laser pulses fired at the slide when held stationary, the stage being moved one step or from the end of one scan line to the beginning of a next scan line on completion of N firings of the laser head. The pulse controller includes a resettable clock circuit which starts running each time it is reset by a detection output pulse from the photomultiplier unit, and only produces an output pulse if it runs to the end of a preset cycling time before the next detection output pulse occurs. Such generation of an output pulse by the resettable clock circuit results in the laser head being caused, after the aforementioned short delay, to fire another laser pulse at the sample slide, and in resetting of the clock circuit. Hence if the firing of a laser pulse fails to result in detection of a fluorescence photon, a subsequent laser pulse is fired after a preset length of time. The preset length of time is preferably controllable by writing a binary number, corresponding to the desired length of time, from a parallel port of the personal computer to a binary register in the clock circuit.
In another preferred embodiment of the invention, the array of sample spots on the slide and the computer controlled stage are replaced by a cuvette filled with liquid sample medium, and a confocal reflector arrangement is employed to direct a large proportion of emitted fluorescence photons into the photomultiplier unit, the focal spot of the objective lens being disposed within the cuvette.
Other preferred features are defined in the dependent claims hereinafter, to which reference should now be made.